Gram negative bacteria secrete a large number of proteins to the extracellular milieu, and many of these proteins are involved in pathogenesis. Of the mechanisms of protein secretion, the autotransporter (AT) pathway is thought to be the most common, yet much remains to be elucidated. This application is a request for continuation of funding of a productive R01 award, the major contributions of which included the discovery of a large groups of secreted gram negative virulence factors (the serine protease autotransporters of Enterobacteriaceae [SPATEs]), the characterization of the modes of action of at least two of these proteins, and the characterization of a novel C-terminal AT domain that catalyzes outer membrane translocation. The present application represents an interdisciplinary effort among pathogenesis, bacteriology, protein biochemistry, and structural biology laboratories. The aims of the application are as follows. Aim 1. Structure-function of SPATE toxins. This aim will address the functions of SPATEs and the structural basis for this diversity. Three inter-related subaims will be pursued: a) Dissection of SPATE structure-function relationships. b) Can spectrin or FAK cleavage be the mechanism of action of Pet?. This subaim will use RNAi technology to assess the role of spectrin and FAK loss in epithelial cells. c) Elucidation of the role of the SPATEs as colonization factors. Aim 2. Autotransporter translocation through the outer membrane. This aim will be pursued as four inter-related subaims: a) Role of the SPATE beta-helix. We will work with Dr. Patricia Clark, who has pioneered the study of the AT beta-helix. b) Characterization of the SPATE AC region. c) Characterization of inter-protein interactions in AT translocation, including the roles of periplasmic chaperones. d) Characterization of intra-protein interactions in AT translocation. This work will extend knowledge on a very common set of virulence factors and elicidate important features of the most common means of outer membrane translocation in gram negative bacteria.